Hi, I'm having trouble with my tranformation. I'm currently trying to do a subclonning with plasmid PBC sk(+), the thing is that I'm having colonies and they are positive in colony PCR but when I extract that DNA and digest it using pvuII it will have antoher pattern appears same as the empty plasmid also digested, so I'm currently trying to figure it out wether is my ligation or something else, cause I believe those are false positives
It may be that your ligation failed but if your pcr primers are both inside the insert sequence then the wetting effect of the ligation mix on the plate will act as a template for the colony pcr.It is better to do colony pcr with one primer in the insert and the other in the plasmid
Did you clone into a selection marker to select your positive clones using the principle of insertional inactivation?
These are likely false positives; your primers are potentially hybridizing with regions found on the empty plasmid or on the genome of your bacteria. To confirm this, perform a PCR on bacteria transformed with an empty plasmid.
The reason for the absence of the insert may be due to either ligation or the plasmid remaining closed because the digestion to open it failed.
Colony PCR very often gives false positives. It seems like your cloning failed, maybe you could share some details about what you did so we could help troubleshoot it.
Yes, I subcloned 2 inserts (400pb) and other 3 of (900pb) in pBC sk(+). I used a molar ratio of 7:1 insert to vector I did it on NEBiocalculator. I digested the plasmid with the restriction enzyme SmaI and purifed my PCR product with (Phusion HF DNA polymerase) by PEPS'Ys protocol;, after this, I tried defosforilating my vector by adding 0.5uL of CIP enzyme also from NEB and let it react at 37°C for 30 min then innactivate for 2 min in 75°C. About the ligation I used 1uL of T4 ligase, 2.5uL of buffer and let it overnight at 25°C. Then I heat inactivate for 65°C for 10 min and did the transformation in competent E.Coli cells. I did these transformation with 5 different inserts and just one was positive in the puvII digestion after plasmid DNA extraction with miniprep from the candidate colonies .I then cloned this last one in pGADT7 for 2H assay and got no colonies at all.
Not sure what's PEPS'Ys protocol. It sounds like you worked with purified PCR products--not digested ones (completely normal for blunt-end cloning). Did you make sure your primers are phosphorylated though?
Well, if you have one positive colony you have the DNA ready for the pGADT7 cloning. So, that's a start. It's not ideal to have low success in cloning but you might have enough to move forward for now. I'd suggest that you sequence that one positive clone first before and after you move it to the final vector.
Ligase buffer does degrade over time, I'd start with a fresh tube if I were you. NEB also sells quick ligase, and they often will send trial size kits if you ask.
Hope this helps!
I will verify the primers I'm using and also sequence the clone. Thank you!
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